Description of <i>Gopheromyces tardescens</i>, gen. nov., sp. nov., <i>Gigasporangiomyces pilosus,</i> gen. nov., sp. nov., <i>Kelyphomyces adhaerens,</i> gen. nov., sp. nov., and proposal of <i>Testudinimycetaceae,</i> fam. nov

Anaerobic gut fungi (AGF, <i>Neocallimastigomycota</i>) represent a phylum of zoospore-producing fungi inhabiting the gastrointestinal tracts of herbivores. Twenty mammalian-affiliated genera (M-AGF) and two tortoise-affiliated genera (T-AGF) have been described so far. Here, we report on three additional novel T-AGF isolates obtained from Texas tortoises (<i>Gopherus berlandieri</i>; GX isolates) and African spurred or sulcata tortoises (<i>Centrochelys sulcata</i>; SR and TM isolates). Phylogenetic analysis using the D1-D2 regions of the large ribosomal RNA subunit (D1-D2 LSU), RNA polymerase II large subunit, internal transcribed spacer-1 (ITS-1) region and transcriptomics-enabled phylogenomic analysis clustered these strains into three distinct, deep-branching clades, closely related to previously described T-AGF genus <i>Testudinimyces</i>. All isolates displayed filamentous rhizoidal growth patterns and produced monoflagellated zoospores. Unique morphological characteristics included the production of elongated, thick, nucleated structures in GX isolates, the formation of thin hair-like projections on sporangial walls in SR isolates and irregularly shaped sporangia in TM isolates. All strains grew optimally at 32-35 °C and showed distinct substrate utilization capacity (e.g. growth on pectin, chitin, galactose). LSU analyses revealed GX isolates as the first cultured representatives of tortoise-affiliated but previously uncultured lineage NY56, while SR and TM strains have not been encountered in prior culture-independent AGF surveys. We propose to accommodate these isolates in three new genera and species - <i>Gopheromyces tardescens</i> (GXA2), <i>Gigasporangiomyces pilosus</i> (SR0.6) and <i>Kelyphomyces adhaerens</i> (TM0.3). Furthermore, based on the ecological, physiological and phylogenetic distinctions between T-AGF and M-AGF, we propose to establish a new family (<i>Testudinimycetaceae</i>) to accommodate the genera <i>Testudinimyces, Gopheromyces</i>, <i>Gigasporangiomyces</i> and <i>Kelyphomyces</i>.